Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Climatic forcing and larval dispersal capabilities shape the replenishment of fishes and their habitat‐forming biota on a tropical coral reef

Fluctuations in marine populations often relate to the supply of recruits by oceanic currents. Variation in these currents is typically driven by large‐scale changes in climate, in particular ENSO (El Nino Southern Oscillation). The dependence on large‐scale climatic changes may, however, be modified by early life history traits of marine taxa. Based on eight years of annual surveys, along 150 km of coastline, we examined how ENSO influenced abundance of juvenile fish, coral spat, and canopy‐forming macroalgae. We then investigated what traits make populations of some fish families more reliant on the ENSO relationship than others. Abundance of juvenile fish and coral recruits was generally positively correlated with the Southern Oscillation Index (SOI), higher densities recorded during La Niña years, when the ENSO‐influenced Leeuwin Current is stronger and sea surface temperature higher. The relationship is typically positive and stronger among fish families with shorter pelagic larval durations and stronger swimming abilities. The relationship is also stronger at sites on the coral back reef, although the strongest of all relationships were among the lethrinids (r = .9), siganids (r = .9), and mullids (r = .8), which recruit to macroalgal meadows in the lagoon. ENSO effects on habitat seem to moderate SOI–juvenile abundance relationship. Macroalgal canopies are higher during La Niña years, providing more favorable habitat for juvenile fish and strengthening the SOI effect on juvenile abundance. Conversely, loss of coral following a La Niña‐related heat wave may have compromised postsettlement survival of coral dependent species, weakening the influence of SOI on their abundance. This assessment of ENSO effects on tropical fish and habitat‐forming biota and how it is mediated by functional ecology improves our ability to predict and manage changes in the replenishment of marine populations.     

Aberrant energy-linked reactions in mitochondria isolated from the livers of rats infected with the liver fluke Fasciola hepatica.

The respiratory properties of mitochondria isolated from the livers of rats infected with the parasite Fasciola hepatica were examined. Oligomycin-sensitive ATPase activity was also examined during the acute stage (2-4 weeks post-infection). At 2,4 and 6 weeks post-infection, mitochondrial respiration in vitro (supported by site I and site II substrates) was completely uncoupled. Limited respiratory control had returned by 11 weeks post-infection, but complete recovery was not observed even at 21 weeks post-infection. At 4 weeks post-infection, uncoupled respiration (from all three energy-conserving sites) was also markedly attenuated (to the greatest extent with NADH-linked substrate). Except for pyruvate-supported respiration, this attenuation was not apparent at any other stage of the infection. The attenuation of pyruvate-supported respiration declined, but was still present, at 6 weeks post-infection. In addition to these perturbations in mitochondrial respiratory properties, mitochondrial ATPase activity at 4 weeks post-infection was insensitive to oligomycin, indicating a change in the structural integrity of the ATPase complex.     

Mapping the substrate-binding site of a human class mu glutathione transferase using nuclear magnetic resonance spectroscopy.

The substrate-binding site of a human muscle class mu glutathione transferase has been characterized using high-resolution nuclear magnetic resonance spectroscopy. Isotopic labeling has been used to simplify one-dimensional proton NMR spectra of the Tyr and His residues in the enzyme and two-dimensional carbon-proton spectra of the Ala and Met residues in the enzyme. The resonance lines from 8 of the 12 Tyr residues have been assigned using site-directed mutagenesis. Replacement of Tyr7 with Phe reduced the activity of the enzyme 100-fold. The proximity of His, Tyr, Ala, and Met residues to the active site has been determined using a nitroxide-labeled substrate analogue. This substrate analogue binds with high affinity (Keq = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 A of the active site. Three of the assigned Tyr residues are greater than 17 A from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 A from the active site. Broadening of the Ala and Met resonance lines by the spin-labeled substrate indicates that three Ala residues are 9-16 A from the nitroxide, three Met residues are less than 9 A from the nitroxide, and two Met residues are 9-16 A from the nitroxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Effect of adipose tissue site, animal size, and fasting on lipolysis in bovine adipose tissue in vitro.

1. Lipolytic rates expressed as mumol glycerol released per mg protein increased with body weight in Holstein steers. 2. Lipolytic rates were greatest in both inner and outer back fat and lowest in omental, perirenal, and intermuscular fat depots. 3. Epinephrine stimulated overall glycerol release 3-5-fold. 4. Fasting resulted in greater basal lipolytic rates but epinephrine-stimulated rates tended to be greater for nonfasted steer adipose tissue. 5. Lipolytic activity in adipose tissue seems to increase with growth and fattening, and differences in lipolytic rates between various depots diminish with growth.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

Procalcitonin Identifies Cell Injury,Not Bacterial Infection,in Acute Liver Failure

Background

Because acute liver failure (ALF) patients share many clinical features with severe sepsis and septic shock, identifying bacterial infection clinically in ALF patients is challenging. Procalcitonin (PCT) has proven to be a useful marker in detecting bacterial infection. We sought to determine whether PCT discriminated between presence and absence of infection in patients with ALF.

Method

Retrospective analysis of data and samples of 115 ALF patients from the United States Acute Liver Failure Study Group randomly selected from 1863 patients were classified for disease severity and ALF etiology. Twenty uninfected chronic liver disease (CLD) subjects served as controls.

Results

Procalcitonin concentrations in most samples were elevated, with median values for all ALF groups near or above a 2.0 ng/mL cut-off that generally indicates severe sepsis. While PCT concentrations increased somewhat with apparent liver injury severity, there were no differences in PCT levels between the pre-defined severity groups–non-SIRS and SIRS groups with no documented infections and Severe Sepsis and Septic Shock groups with documented infections, (p = 0.169). PCT values from CLD patients differed from all ALF groups (median CLD PCT value 0.104 ng/mL, (p ≤0.001)). Subjects with acetaminophen (APAP) toxicity, many without evidence of infection, demonstrated median PCT >2.0 ng/mL, regardless of SIRS features, while some culture positive subjects had PCT values <2.0 ng/mL.

Summary/Conclusions

While PCT appears to be a robust assay for detecting bacterial infection in the general population, there was poor discrimination between ALF patients with or without bacterial infection presumably because of the massive inflammation observed. Severe hepatocyte necrosis with inflammation results in elevated PCT levels, rendering this biomarker unreliable in the ALF setting.     

Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers

Background

Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined.

Methods

In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)₂D, 24,25-dihydroxyvitamin D (24,25(OH)₂D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls.

Results

Serum Ca and 1,25(OH)₂D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)₂D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)₂D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)₂D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)₂D, were not observed. 1,25(OH)₂D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05).

Conclusions

Quantitative differences in serum Ca and 1,25(OH)₂D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.